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Mol Nutr Food Res. 2019 Sep;63(18):e1900336. doi: 10.1002/mnfr.201900336. Epub 2019 Jun 26.

Rational Design, Structure-Activity Relationship, and Immunogenicity of Hypoallergenic Pru p 3 Variants.

Author information

1
Department of Biosciences, University of Salzburg, Hellbrunnerstraße 34, 5020, Salzburg, Austria.
2
Allergy Clinic, Dept. 22, Herlev-Gentofte Hospital, Kildegaardsvej 28, 2900, Hellerup, Denmark.
3
Department of Experimental Immunology, Amsterdam University Medical Centers, Meibergdreef 9, 1105AZ, Amsterdam, The Netherlands.
4
Food & Health Programme, Inst. of Food Research, Norwich, Norfolk, NR4 7UQ, United Kingdom.
5
R&D Department, ROXALL Group, Parque Científico y Tecnológico de Bizkaia, Edif. 401, 48170, Zamudio, Spain.
6
Department of Clinical Pharmacology, Hospital Clinico San Carlos, c/ Prof. Martín Lagos s/n, 28040, Madrid, Spain.
7
Department of Allergy, Hospital Clinico San Carlos, c/ Prof. Martín Lagos s/n, 28040, Madrid, Spain.
8
Division of Infection, Immunity & Respiratory Medicine, University of Manchester, Royal Manchester Children's Hospital, Manchester, M13 9WL, United Kingdom.
9
Allergy Dpt, 2nd Pediatric Clinic, University of Athens, 41, Fidippidou, Athens, 115 27, Greece.
10
Center of Molecular Allergology, IDI, Via dei Monti di Creta 104, ZIP 00167, Rome, Italy.
11
Associated Centers for Molecular Allergology, Via Portuense 700, ZIP 00149, Rome, Italy.
12
Department of Otorhinolaryngology, Amsterdam University Medical Centers, Meibergdreef 9, 1105AZ, Amsterdam, The Netherlands.

Abstract

SCOPE:

Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy.

METHODS AND RESULTS:

Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model.

CONCLUSIONS:

An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

KEYWORDS:

Pru p 3; allergen immunotherapy; hypoallergens; lipid transfer proteins; peach allergies

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